Maize Transformation Research Articles

Genetic transformation of tropical maize (Zea mays L.) inbred line with a phytase gene from Aspergillus niger.

A full-length cDNA of phyA gene of Aspergillus niger, encoding phytase enzyme, was cloned and expressed in E. coli BL21 cells and assayed for its activity. The phyA cDNA consisted of 1404bp, which encoded 467 amino acid residues. The phytase activity of purified phytase was 826.33U/mL. The phyA gene under the control of endosperm-specific promoters was transformed into an Indian maize inbred line, UMI29, using particle bombardment-mediated transformation method to generate transgenic maize plants over-expressing phytase in seeds. PCR and GUS analyses demonstrated the presence of transgenes in T0 transgenic plants and their stable inheritance in the T1 progenies. Three transgenic events expressing detectable level of A. niger phytase were characterized by western blot analysis. Phytase activity of 463.158U/kg of seed was observed in one of the events, JB-UMI29-Z17/2. The phytase activity of transgenic maize seeds was 5.5- to 7-fold higher than the wild-type UMI29 seeds and, consequently, the seeds had 0.6- to 5-fold higher inorganic phosphorus content.

A study on optimization of pat gene expression cassette for maize transformation.

Phosphinothricin acetyltransferase gene (pat) is an important selectable marker and also a key herbicide trait gene in several commercial products. In maize, the transformation frequency (TF) using pat as a selectable marker is the lowest among the commonly used marker options including epsps, pmi or ppo. Low pat transformation efficiency can become a major bottleneck in our ability to efficiently produce large numbers of events, especially for large molecular stack vectors with multiple trait gene cassettes. The root cause of the lower efficiency of pat in maize is not well understood and it is possible that the causes are multifaceted, including maize genotype, pat marker cassette, trait gene combinations and selection system. In this work we have identified a new variant of pat gene through codon optimization that consistently produced a higher transformation frequency (> 2x) than an old version of the pat gene that has codons optimized for expression in dicot plants. The level of PAT protein in all 16 constructs was also found multifold higher (up to 40 fold) over that of the controls. All of the T0 low copy transgenic plants generated from the 16 different constructs showed excellent tolerance to ammonium glufosinate herbicide spray tests at 4x and 8x recommended field application rates (1x = 595g active ingredient (ai)/hectare of ammonium glufosinate) in the greenhouse.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins.

Inspired by Homer´s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize.

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell's genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR.

Infection of Embryonic Callus with Agrobacterium Enables High-Speed Transformation of Maize.

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium-mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T0 generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.

Evaluation of the Genotype-dependency of the Leafbased Regeneration and Transformation System in Maize

Tissue culture and genetic transformation in maize are very laborious. The existing regeneration methods, which mainly use immature embryos as starting material, are highly genotype-dependent. Leaf segments can be used as an alternative explant source to produce embryogenic calli. Although a reliable leafbased regeneration and transformation system has been recently reported for maize, however, the genotype-dependency of this method has not been described yet. To this end, we evaluated the production of embryogenic calli from young leaves of several maize genotypes. The results showed that, overall callus induction potential as well as embryogenic callus induction rate is significantly different among the tested genotypes, demonstrating the genotypedependency of this system. However, induced embryogenic calli from different genotypes remained their embryogenic capability during several callus multiplication rounds. In addition, embryogenic calli showed high potential for biolistic-based genetic transformation, as revealed by transient GUS expression.
 Plant Tissue Cult. & Biotech. 28(2): 261-268, 2018 (December)

Maize Transformation Using the Morphogenic Genes Baby Boom and Wuschel2.

Despite the fact that maize transformation has been available for over 25 years, the technology has remained too specialized, labor-intensive, and inefficient to be useful for the majority of academic labs. Compounding this problem, future demands in maize genome engineering will likely require a step change beyond what researchers view as "traditional" maize transformation methods. Recently, we published on our use of constitutively expressed morphogenic transcription factors Baby Boom (Bbm) and Wuschel2 (Wus2) to improve maize transformation, which requires CRE-mediated excision before regeneration of healthy, fertile T0 plants. Moving beyond this first-generation system, we have developed a new expression system for Bbm and Wus2, using a non-constitutive maize phospholipid transferase protein promoter (Pltp pro) driving Bbm expression and a maize auxin-inducible promoter (Axig1 pro) for WUS2 expression. Using this combination of expression cassettes, abundant somatic embryos rapidly form on the scutella of Agrobacterium-transformed zygotic immature embryos. These somatic embryos are uniformly transformed and can be directly germinated into plants without a callus phase. Transformed plants are sent to the greenhouse in as little as 1 month, and these T0 plants match the seed-derived phenotype for the inbred and are fertile. T1 seeds germinate normally and have a uniformly wild-type inbred phenotype. This new system represents a rapid, user-friendly transformation process that can potentially facilitate high-throughput production of transgenic T0 plants in B73, Mo17, and the recently developed Fast-Flowering Mini-Maize.

Genetic Dissection of Maize Embryonic Callus Regenerative Capacity Using Multi-Locus Genome-Wide Association Studies.

The regenerative capacity of the embryonic callus, a complex quantitative trait, is one of the main limiting factors for maize transformation. This trait was decomposed into five traits, namely, green callus rate (GCR), callus differentiating rate (CDR), callus plantlet number (CPN), callus rooting rate (CRR), and callus browning rate (CBR). To dissect the genetic foundation of maize transformation, in this study multi-locus genome-wide association studies (GWAS) for the five traits were performed in a population of 144 inbred lines genotyped with 43,427 SNPs. Using the phenotypic values in three environments and best linear unbiased prediction (BLUP) values, as a result, a total of 127, 56, 160, and 130 significant quantitative trait nucleotides (QTNs) were identified by mrMLM, FASTmrEMMA, ISIS EM-BLASSO, and pLARmEB, respectively. Of these QTNs, 63 QTNs were commonly detected, including 15 across multiple environments and 58 across multiple methods. Allele distribution analysis showed that the proportion of superior alleles for 36 QTNs was <50% in 31 elite inbred lines. Meanwhile, these superior alleles had obviously additive effect on the regenerative capacity. This indicates that the regenerative capacity-related traits can be improved by proper integration of the superior alleles using marker-assisted selection. Moreover, a total of 40 candidate genes were found based on these common QTNs. Some annotated genes were previously reported to relate with auxin transport, cell fate, seed germination, or embryo development, especially, GRMZM2G108933 (WOX2) was found to promote maize transgenic embryonic callus regeneration. These identified candidate genes will contribute to a further understanding of the genetic foundation of maize embryonic callus regeneration.

An improved ternary vector system for Agrobacterium-mediated rapid maize transformation

Key messageA simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation.The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86–103%) raw transformation frequency in an elite maize inbred.

Effect of harmones on callus induction in Maize (Zea mays L.)

Callus induction from explants is a critical process in regeneration, micropropagation and transformation of maize (Zea mays L.) plants. Formation of callus from plant tissues on culture is affected by several factors. This study revealed to establish the effect of genotype, source of explants and auxin concentration on callus induction from five genotypes UMI 757 (G1), UMI 615 (G2), UMI 112 (G3), UMI 285 (G4) and CO 1 (G5) and one hybrid CO H (M) 5 (G6). Callus induction of the six maize varieties was investigated using immature embryos (E1), leaf bits (E2), root tips (E3), hypocotyls (E4) and seeds (E5) as explants with different concentrations of hormones. In this study, immature embryo was taken from 10 to 12 days after pollination (DAP) to get maximum response. The highest percentage of callus induction was observed (99.10) in immature embryo culture and seed culture gave the highest percentage of rhizogenic callus formation when compare to immature embryo. Among the genotypes tested, CO H (M) 5 recorded the highest callus induction percentage on (2D2K2) medium composition.

Transcriptome Analysis of Maize Immature Embryos Reveals the Roles of Cysteine in Improving Agrobacterium Infection Efficiency.

Maize Agrobacterium-mediated transformation efficiency has been greatly improved in recent years. Antioxidants, such as, cysteine, can significantly improve maize transformation frequency through improving the Agrobacterium infection efficiency. However, the mechanism underlying the transformation improvement after cysteine exposure has not been elucidated. In this study, we showed that the addition of cysteine to the co-cultivation medium significantly increased the Agrobacterium infection efficiency of hybrid HiII and inbred line Z31 maize embryos. Reactive oxygen species contents were higher in embryos treated with cysteine than that without cysteine. We further investigated the mechanism behind cysteine-related infection efficiency increase using transcriptome analysis. The results showed that the cysteine treatment up-regulated 939 genes and down-regulated 549 genes in both Z31 and HiII. Additionally, more differentially expressed genes were found in HiII embryos than those in Z31 embryos, suggesting that HiII was more sensitive to the cysteine treatment than Z31. GO analysis showed that the up-regulated genes were mainly involved in the oxidation reduction process. The up-regulation of these genes could help maize embryos to cope with the oxidative stress stimulated by Agrobacterium infection. The down-regulated genes were mainly involved in the cell wall and membrane metabolism, such as, aquaporin and expansin genes. Decreased expression of these cell wall integrity genes could loosen the cell wall, thereby improving the entry of Agrobacterium into plant cells. This study offers insight into the role of cysteine in improving Agrobacterium-mediated transformation of maize immature embryos.

Advances in Agrobacterium-mediated Maize Transformation.

One of the major limitations of maize transformation is the isolation of a large number of immature embryos using the time-consuming manual extraction method. In this article, we describe a novel bulk embryo extraction method for fast isolation of a large number of embryos suitable for both biolistic- and Agrobacterium-mediated transformation. Optimal gene delivery and tissue culture conditions are also described for achieving high efficiency in Agrobacterium-mediated maize transformation using phosphomannose isomerase (PMI) as a selectable marker.

Agrobiodiversidad, género y soberanía alimentaria en Tlaxiaco, Oaxaca

El maíz ha sido la base de la alimentación de los pueblos mesoamericanos por milenios. En México existen 59 razas de maíz y una gran variedad de platillos que incluyen al grano. Históricamente las mujeres han sido las responsables de su preparación, siendo la tortilla la base de muchos de ellos. Este artículo analiza las prácticas de manejo de maíz que realizan las mujeres mixtecas que integran la Unión de Palmeadoras de Tlaxiaco, Oaxaca. Las mujeres se dedican a la elaboración y venta diaria de tortilla artesanal. Los datos se obtuvieron en 2015 y 2016 a través de una estrategia metodológica que involucró entrevistas, talleres, observación participante, diarios de campo y un censo. Se describen las redes de abastecimiento de maíz, su proceso de nixtamalización, y las prácticas de comercialización de las mujeres. Los resultados muestran que las palmeadoras promueven el comercio de maíces en la región, poseen conocimientos relacionados con su transformación en alimento, y contribuyen a la diversidad nutricional de la población con la venta de tortillas hechas a mano con varios tipos de maíz. Las mujeres son pieza clave en la conservación in situ del maíz criollo, ya que promueven el consumo de maíces locales y estimulan su producción. Sus habilidades y necesidades deben ser tomadas en cuenta en políticas públicas encaminadas a la soberanía alimentaria de México.

Integrative analysis of DNA methylation, mRNAs, and small RNAs during maize embryo dedifferentiation

BackgroundMaize (Zea mays) is an important model crop for transgenic studies. However, genetic transformation of maize requires embryonic calli derived from immature embryo, and the impact of utilizing tissue culture methods on the maize epigenome is poorly understood. Here, we generated whole-genome MeDIP-seq data examining DNA methylation in dedifferentiated and normal immature maize embryos.ResultsWe observed that most of the dedifferentiated embryos exhibited a methylation increase compared to normal embryos. Increased methylation at promoters was associated with down-regulated protein-coding gene expression; however, the correlation was not strong. Analysis of the callus and immature embryos indicated that the methylation increase was induced during induction of embryonic callus, suggesting phenotypic consequences may be caused by perturbations in genomic DNA methylation levels. The correlation between the 21-24nt small RNAs and DNA methylation regions were investigated but only a statistically significant correlation for 24nt small RNAs was observed.ConclusionsThese data extend the significance of epigenetic changes during maize embryo callus formation, and the methylation changes might explain some of the observed embryonic callus variation in callus formation.

Agrobacterium-mediated genetic transformation of a tropical elite maize line

Abstract: The efficiency of maize transformation mediated by Agrobacterium tumefaciens is influenced by various factors. The aim of this study was to test the effect of different concentrations of N6 salts (50% – half strength, and 100% - full strength) in the infection and cocultivation media on genetic trans-formation efficiency of the L3 tropical elite maize line. Immature embryos were transformed via A. tumefaciens harboring the binary vector pTF102 containing the genes uidA and BAR under control of the CaMV35S promoter. The efficiency of the transgenic events produced was 3% for full strength and 1.1% for half strength N6 salts. Although under a lower concentration of salts, GUS expres-sion was stronger; under this concentration, regeneration was less efficient. Thus, the results showed that the presence of 100% N6 salts in the infection and cocultivation media favored genetic transformation of the L3 maize inbred line mediated by A. tumefaciens. Key words : Immature embryos, salt concentration, somatic embryogenesis, Zea mays.

Agrobacterium tumefaciens-MEDIATED IN-PLANTA TRANSFORMATION OF INDONESIAN MAIZE USING pIG121Hm-Cs PLASMID CONTAINING nptII AND hpt GENES

&lt;p class="abstrakInggris"&gt;&lt;span&gt;Maize (&lt;em&gt;Zea mays&lt;/em&gt; L.) productivity in Indonesia is challenged to be increased using genetic engineering. Recent advances in &lt;em&gt;Agrobacterium tumefaciens&lt;/em&gt;-mediated &lt;em&gt;in-planta&lt;/em&gt; transforma-tion makes it possible&lt;strong&gt; &lt;/strong&gt;to&lt;strong&gt; &lt;/strong&gt;transform maize with low cost, and simple method. This study aimed to confirm pIG121Hm-&lt;em&gt;Cs&lt;/em&gt; plasmid in &lt;em&gt;A. tumefaciens&lt;/em&gt;, and to estimate the efficiency level of&lt;strong&gt; &lt;/strong&gt; &lt;em&gt;A&lt;strong&gt;. &lt;/strong&gt;tumefaciens&lt;/em&gt;-mediated &lt;em&gt;in-planta&lt;/em&gt; transformation of Indonesian maize by&lt;strong&gt; &lt;/strong&gt;using pIG121Hm-&lt;em&gt;Cs&lt;/em&gt; plasmid containing &lt;em&gt;npt&lt;/em&gt;II and&lt;strong&gt; &lt;/strong&gt;&lt;em&gt;hpt&lt;/em&gt;&lt;strong&gt; &lt;/strong&gt;genes. A series of studies were conducted including confirmation of gene construct of pIG121Hm-&lt;em&gt;Cs&lt;/em&gt; plasmid in &lt;em&gt;A. tumefaciens&lt;/em&gt;, transformation of four maize lines through &lt;em&gt;A. tumefaciens&lt;/em&gt;-mediated &lt;em&gt;in-planta&lt;/em&gt; technique, acclimatization of transformant plants and molecular analysis of selected plants using polymerase chain reaction (PCR). The pIG121Hm-&lt;em&gt;Cs&lt;/em&gt; plasmid was confirmed via PCR analysis using specific primers of &lt;em&gt;npt&lt;/em&gt;II and &lt;em&gt;hpt&lt;/em&gt; genes and resulted 700 bp and 500 bp for fragments of &lt;em&gt;npt&lt;/em&gt;II and &lt;em&gt;hpt&lt;/em&gt;, respectively. After selection, acclimatization and molecular analysis steps, the efficiency levels of transformation of four maize lines were low, ranging from 3.8% to 12.8%. The level of transformation efficiency of ST-27 line was the highest accounting for 12.8% of 45 planted embryos on selection medium based on PCR analysis using specific primer for &lt;em&gt;npt&lt;/em&gt;II gene. Overall, &lt;em&gt;A. tumefaciens&lt;/em&gt;-mediated &lt;em&gt;in planta&lt;/em&gt; transformation on maize floral pistil in this study proved to be successful and rapid. Therefore, this enhanced transformation method will be beneficial for Indonesian maize genetic engineering.&lt;/span&gt;&lt;/p&gt;

Comparison of the impact of viral and plant-derived promoters regulating selectable marker gene on maize transformation and transgene expression

Key messageThe choice of promoter regulating the selectable marker gene impacts transformation efficiency, copy number and the expression of selectable marker and flanking genes in maize.Viral or plant-derived constitutive promoters are often used to regulate selectable marker genes. We compared two viral promoters, cauliflower mosaic virus (CaMV 35T) and sugarcane bacilliform virus (SCBV) with two plant promoters, rice actin1 (OsAct1) and maize ubiquitin 1 (ZmUbi1) to drive aryloxyalkanoate dioxygenase (aad-1) selectable marker gene in maize inbred line B104. ZmUbi1- and OsAct1-containing constructs demonstrated higher transformation frequencies (43.8 and 41.4%, respectively) than the two viral promoter constructs, CaMV 35T (25%) and SCBV (8%). Interestingly, a higher percentage of single copy events were recovered for SCBV (82.1%) and CaMV 35T (59.3%) promoter constructs, compared to the two plant-derived promoters, OsAct1 (40.0%), and ZmUbi1 (27.6%). Analysis of protein expression suggested that the viral promoter CaMV 35T expressed significantly higher AAD-1 protein (174.6 ng/cm2) than the OsAct1 promoter (12.6 ng/cm2) in T0 leaf tissue. When measured in T2 callus tissue, the two viral promoters both had higher expression and more variability than the two plant-derived promoters. A potential explanation for why viral promoters produce lower transformation efficiencies but higher percentages of low copy number events is discussed. In addition, viral promoters regulating aad-1 were found to influence the expression of upstream flanking genes in both T0 leaf and T2 callus tissue.

Advances in Maize Transformation Technologies and Development of Transgenic Maize.

Maize is the principal grain crop of the world. It is also the crop where genetic engineering has been employed to a great extent to improve its various traits. The ability to transform maize is a crucial step for application of gene technology in maize improvement. There have been constant improvements in the maize transformation technologies over past several years. The choice of genotype and the explant material to initiate transformation and the different types of media to be used in various stages of tissue culture can have significant impact on the outcomes of the transformation efforts. Various methods of gene transfer, like the particle bombardment, protoplast transformation, Agrobacterium-mediated, in planta transformation, etc., have been tried and improved over years. Similarly, various selection systems for retrieval of the transformants have been attempted. The commercial success of maize transformation and transgenic development is unmatched by any other crop so far. Maize transformation with newer gene editing technologies is opening up a fresh dimension in transformation protocols and work-flows. This review captures the various past and recent facets in improvement in maize transformation technologies and attempts to present a comprehensive updated picture of the current state of the art in this area.

Characterization of transformations of maize residues into soil organic matter.

An awareness of the transformation of plant residues returned to cultivated soils is vital for a better understanding of carbon cycles, the maintenance of soil fertility and the practice of a sustainable agriculture. The transformation of maize (Zea mays L) straw residues into soil organic matter (SOM) in a one year incubation experiment was studied in a soil that had been under long term cultivation with wheat (Triticum aestivum L) for >30years. A novel sequential exhaustive extraction and fractionation procedure isolated a series of fractions of SOM. The samples were characterized by elemental and δ13C analyses, by amino acids and neutral sugars analyses, by Fourier transformed infrared (FTIR) spectrometry, and by solid state 13C nuclear magnetic resonance (NMR) spectroscopy and with chemical shift anisotropy (CSA) -filter and dipolar dephasing (DD) spectral editing NMR techniques. The δ13C data indicated that 59% and 38% of the newly transformed organic carbon was in the humic and fulvic acid fractions, respectively, and in general a greater proportion of the transformed carbon was in the fractions isolated at the higher pH values. Results for SOM fractions from the amended soil indicate dominant contributions from carbohydrate and lignin-like material, and that can be clearly identified by FTIR, CP/TOSS, and spectral editing of CSA-filter and DD. The compositions of the fractions from the amended and non-amended soils fractions can be clearly differentiated using principal component analysis (PCA) for the data collected. The sequential extraction procedure showed that the hydrophilicity of humic fractions increased as the result of the maize amendment, and the aromaticity of the fraction decreased. The data may give some indications of transformations that take place during humification processes.

An efficient regeneration and genetic transformation of maize through Agrobacterium and particle bombardment in immature embryos

An efficient Agrobacterium mediated transformation system, from which transgenic tropical maize plants was directly generated without previous crosses with laboratory or temperate lines was established in present study. Experimental evaluations are focused on two main issues: i) Establishment of appropriate tissue culture conditions, which induced somatic embryogenesis from the immature embryo cells, and ii) The delivery of T-DNA toward these cells. A high rate of embryogenic calli, generated from immature embryo was obtained when 2-4 D (4mgL-1) was added to the MS based induction medium. Regeneration rate was observed upto 19 plants per gram in the calli by using BAP and KIN at 0.5mgL-1. Regarding the Agrobacterium strains evaluated for their transformation capability on the tropical maize line MU-2092 used, best results are obtained from the strain when applied at OD 550nm= 0.5-1.0. Physical micro-wounds before the Agro-infection proved to be an excellent way to promote both the T-DNA transfer toward the immature embryo and the acceleration in rates of transient GUS expression when compared with particle bombardment. The highest frequencies of transient GUS expression corresponding to the embryos as well as the regeneration of whole transgenic plants emerged from them, was obtained using immature embryos wounded by bombarding at 80 lb/in2. Analysis of the progenies confirmed the sexual transmission of the introduced genes and their stable expression.